Isolation of bacteria



United States Patent 3,072,538 ISOLATION OF BACTERIA James N. Baptist, Laurel, Md., assignor to W. R. Grace 8: Co., New York, N .Y., a corporation of Connecticut No Drawing. Filed Dec. 30, 1960, Ser. No. 79,488

18 Claims. (Cl. 195-101) This invention is concerned with the isolation of strains of bacteria which are active producers of poly-fl-hydroxybutyric acid. 1

In summary, thi is a method of separating strains of living baceteria capable of synthesizing poly-p-hydroxybutyric acid from mixtures thereof with other strains contained in aqueous suspension which comprises adding a layer of said suspension on top of an inert sterile solution having a density greater than that of the said suspension, centrifuging the resulting combination whereby the cells of bacteria capable of producing said polyester in high yield sink into said solution and recovering at least a portion of said solution.

Generally in carrying out this invention as illustrated in the examples, a mixed bacterial suspension is first prepared. The bacterial suspension is then treated with a nutrient to promote poly-,B-hydroxybutyric acid synthesis by those bacteria capable of synthesizing this polyester. .A sterile high density solution, suitably a 60% sucrose solution, is added to a sterile centrifuge tube. The density of the high density solution can vary from about 1.1 to 1.3. The concentration of the sucrose solutiontherefore can vary from about 25 to 60%. Other sugars which can be used to prepare the high density solution include, for example, glucose, fructose, maltose and trehalosa salts sufiiciently soluble in water to give the right density ranges and which are non-toxic to bacteria can also be used. These salts include most potassium and sodium salts in addition to such salts as magnesium sulfate, ferric chloride and ammonium sulfate. It is obvious to one skilled in the art that salts of heavy metal ions like silver and mercury are generally toxic to bacteria and could not be used. Most suitable high density solutions are obtained from water soluble high polymers such as polyethylene glycol. High density nonaqueous solutions can be used a long as they are nontoxic to bacteria and have a density within the above mentioned range. The bacterial suspension is added on top of the high density solution as a separate layer and the tube is then centrifuged. During centrifugation the strains of bacteria which are the most active producers of pbly-B-hydroxybutyrit: acid migrate into the lower high density layer.

After centrifugation, a sample of the high density solution is obtained in any of a number of ways without mixing it with the layer above. In one embodiment of this invention the upper layer is siphoned oil? in order to then obtain a sample of the high density solution. In another embodiment of this invention a permanent siphon is attached to a centrifuge tube. The siphon, a tube of small diameter and about the length of the centrifuge tube, is fused onto the inside of the centrifuge tube. A rubber cap is used to plug the top of the siphon, and after centrifugation, the cap is pierced with a narrow syringe needle to withdraw a sample of the high density solution. The preferred embodiment of this invention, however, is to use a plastic centrifuge tube. In this embodiment, after centrifugation, the plastic, tube is punctured with a syringe needle and a sample of the high density solution is withdrawn.

The sample is streaked on conventional solid nutrient media to further bacterial growth. Isolated colonies of bacteria are eventually picked out and microscopically examined to determine the purity of the colony and the presence of lipid-like bodies in the cell. Dried, stained Patented Jan. 8, 1963 slides are employed in all the examples of this invention since staining is useful in revealing structures within the cell. The particular staining method used in this instance was that of K. L. Burdon, J. Bact. 52, p. 665 (1946). This method produces pink stained cells with fat or lipid-like bodies stained blue.

It is obvious that aseptic techniques must be used throughout any process for isolating bacteria. All nutrient media must be sterilized before being employed for cultivation of pure cultures. The media must also be handled in such a way as to prevent the entrance of bacteria other than those under cultivation.

To test for poly-[i-hydroxybutyric acid (polyester) content, the isolated strains are treated with the chosen substrate to further promote polyester synthesis. The polyester then can be removed from the cells by several methods. The preferred method, and the one used throughout this invention, is described in detail in my copending application S.N. 58,154, filed September 26, 1960. As therein described, the bacteria are collected by known means, e.g., centrifugation, and the mass of Wet cells is dried by suitable means, for example, by dispersing them in acetone. Upon removal of the acetone, the bacterial residue is easily dried to a powder. The powder is then treated with pyridine at reflux for 5-30 minutes to dissolve the poly-B-hydroxybutyric acid. The resulting polyester solution is filtered and ether is added to the filtrate to precipitate the polyester.

Poly-fl-hydroxybutyric acid produced by the bacteria isolated by the novel method of this invention and processed as described aforesaid is a translucent, plastic-like polyester which can be utilized in several ways. It is easily cast into a film or molded into articles by conventional methods. It is also useful for surface coatings and as a fiber. It is especially useful in the field of medicine. Medical sutures made of poly-fl-hydroxybutyric acid need not be removed since they eventually decompose to naturally occuring substances without harm to the patient. Films of poly-fl-hydroxybutyric acid can be used to support injured arteries and blood vessels until the tissues heal.

Since in most instances in the following examples the volumes of cultures were small, the yield of polyester was too small to filter and was judged by the turbidity of the final solution as compared with known standards.

Example I A nitrogen free mineral solution having the following compositions was prepared:

20SOPg01:S-%l2ag'e buffer: 46.0 g. KH2PO4, 86.4 g. Na2HPO-7HaO,

\ The mixture was adjusted to between pH 4 and 5 with dilute HCl, autoclaved and allowed to cool.

A sample of soil served as a source of bacteria. The soil sample per se is not critical. It can be obtained from any area and will show substantially the same behavior in this invention. 50 ml. of the nitrogen free mineral solution were mixed with a 5 g. sample of soil and the larger particles were allowed to settle. 40 ml. of the turbid supernatant were added drop-wise as a separate layer to the top of ml. of a 20% sterilizedsucrose solution colored, pink with safranin dye to distinguish the two layers. It is evident that any dye soluble in the solution to be colored can be used. After about 45 minutes most of the remaining particles of soil sank into the sucrose layer leaving a colorless top layer of mixed soil bacteria.

To promote poly-p-hydroxybutyric acid synthesis by those bacteria capable of synthesizing this polyester, 10 ml. of the top layer were pipetted off and added to a 500 ml. Erlenmeyer flask containing 0.2 g. glucose. The flask was incubated overnight at 30 C.

Next, 10 ml. of a sterile 20% sucrose solution colored pink with safranin was added to a sterile plastic centrifuge tube. The contents of the aforesaid flask (about 10 ml.) were carefully added on top of the sucrose solution as a separate layer. The tube was centrifuged 10 minutes on a conventional laboratory centrifuge at about 512,000 ft./sec. After centrifugation, the top layer and part of the sucrose layer were siphoned off with several sterilized pipettes used in series, while avoiding contamination of the lower sucrose layer with any bacteria from the upper layer. A little of the remaining sucrose lagyler was; streaked on an agar medium in Petri dishes.

After 4 days, eight bacteria colonies were picked out and examined under the microscope. Stained slides were made by the aforementioned method. All of the colonies examined looked like pure strains and all had blue spots in the cells.

Each of the eight bacteria varieties, labeled A through H, was grown in 50 ml. of medium prepared according to the following formula:

Glucose g.... 10.0 Mineral solution* ml 25.0 Yeast extract g 0.05 Water mll 500.0

Mineral solution: 10.0 g. (NH4)2SO4, 10.0 g. KHgPOr, 18.9 g. NaQI-IPOI-TH O, 2.0 g. Mg'SOt, 0.2 g. CaClz, 0.06 g. FeCls, 1000.0 ml. H2O.

Strain No. Poly-,8-hydroxybutyric acid yield A Good B Poor C None D None E None F None H Poor The technique was therefore successful in isolating three active polyester producing strains from a mixture that also contained a multitude of inactive strains. Varieties C, D, E and F may have come from cells with a high content of some other dense material or from cells stuck to clay particles.

Example 11 A mixed bacterial suspension was prepared by shaking a 5 gxsoil sample with 50 ml. of the nitrogen free mineral solution described in Example I. After the larger soil particles had settled, two 20 ml. portions of the cloudy supernatant were pipetted oif. To promote polyester synthesis, one sample (sample I) was added to 0.4 g. by weight of ethanol and the second sample (sample II) was added to 0.6 g. sucrose. Both samples were incubated at 30 C. for two hours.

Next, 3.0 ml. of a sterilized 60% aqueous sucrose solution dyed pink with safranin Were added to each of two conical 15 ml. plastic sterilized centrifuge tubes. 3.0 ml. of a sterilized colorless 25% sucrose solution were then aded dropwise to each tube'as a separate layer on top of the 60% sucrose layer. Both bacterial suspensions (samples I and II) were dyed pink with safranin and 3 ml. of each suspension were added to each tube as a separate top layer. Each tube, therefore, contained three layers of solution with the top "and bottom layer dyed pink. Both tubes were centrifuged for 15 minutes at about 320,000 ft./sec.

After centrifugation an aseptic technique was used to remove samples from the respective bottom layers, as will now be described. The outside of each centrifuge tube was rinsed with acetone. A sterilized syringe was-used to puncture a hole through the lower end of the tube and a small sample, about 0.2 ml., of the 60% sucrose solution from each tube was withdrawn. The sample derived from the tube in which sample I was placed is herein called sample Ia, and that from the sample II tube is sample Ila; Since it was not known how many varieties of bacteria would be found in the 60% sucrose layers, both samples Ia and Ila were plated on an agar medium in a number of Petri dishes at different concentrations and incubated at about 30 C. to allow the cells to grow.

After three days a number of well separated colonies were picked out and examined as in Example I. Since it is known that those strains of bacteria that give blue spots by this method of staining are also apt to be active polyester producers, those strains which contained unusually large and intense blue spots were chosen for further testing. These bacteria are described below:

Strain No. Source, Description Sample B 11a Medium rods (sharp blue spots). 0 Ha Medium to big rods (}4 full 0! blue spots). Like B. Small; cells (1 blue spot per cell).

Do. Medium small rods (about 50% blue). Like K (perhaps smaller). Medium fat rods (many intense blue Strains B, C and G were obtained from the sucrose preincubation sample and the rest of the strains were from the ethanol pro-incubation sample.

The strains listed were then grown in 50 ml. batches of an ethanol medium having the following composition:

Ethanol g 20.0 Mineral solution (composition given in Example I) ml 50.0 Yeast extract g 0.5 Water ..ml 1000.0

After four days at 30 C. without agitation, each of the cultures was harvested by centrifugation and tested for polyester content in the same manner as in Example I. The amount of product was estimated by the amount of turbidity and the results were as follows:

Strain No.-- Poly-fl-hydroxybutyric acid yield B Poor C Good G Poor H 0 I O J Poor K Poor L O M Poor 0 Poor Q Good Strains B, C, G, K, L, M, O, and Q were also grown 1n 50ml. batches of sucrose medium having the following composition:

Sucrose g 30.0 Mineral solution (composition given in Example I) 1n1 50.0 Yield extract g 0.5 Glycerine g 5.0 Water ml 1000.0

5 After3 days at 30 C. without agitation, each of the cultures was harvested by centrifugation and assayed for polyester as above with the following results:

Strain Poly-p-hydroxybutyric acid yield B Good. Good (7.1 mg). G Poor. K Poor. L Poor. M 0. Q Poor of the nitrogen free mineral solution disclosed in Example I with a g. soil sample. 1 g. of sucrose was added to the medium which was then incubated at 30 C. for

three hours to promote polyester synthesis.

Next, 5 ml. of a sterilized 60% sucrose solution dyed pink with safranin were added to each of two conical sterilized 15 ml. plastic centrifuge tubes. Two 4 ml. portions of the cloudy supernatant bacterial suspension were siphoned off with sterilized pipettes and added to each centrifuge tube dropwise as a separate layer. Each tube was centrifuged for minutes at about 320,000 ft./sec.

The procedures described in Example II were followed for aseptically removing samples of the 60% sucrose solution as well as for plating the samples on an agar medium in Petri dishes.

After three days a number of well separated colonies were picked out and examined as'in the previous examples. These bacteria are described below:

Strain No.-- Description S-4-l Very big fat rods like sausages. S-4-2 Like S-4-1 but slightly smaller. S4-3 Big rods growing in long chains with bluish black spots.

S4-4 Big rods with big bluish black spots.

S-45 Big rods with a few bluish black spots.

S-46 'Srnall thin rods with blue spots.

S-4-7 Medium sized rods with numerous bluish black spots.

S-48 Small rods with many bluish black spots.

S-4-9 Smaller rods than 5-4-8 with bluish black spots.

spots.

A' few of the strains listed above were discarded since they had not grown as well as the others. The rest of the strains (those noted below) were grown in 50 ml. batches of the following medium:

Glucose g 20.0 Mineral solution -ml 25.0 Yeast extract g 0.25 Water ml 500 After 3 days at 30 C. without agitation, some of the cultures were harvested by centrifugation and tested for polyester content in the same manner as in the previous examples. Some of the strains did not grow on the test medium. Of those strains which did grow twelve were assayed for polyester and only one of these strains did not yield polyester. The others produced varying amounts of polyester product. The amount of product was estimated by the turbidity except in one instance where there was enough product to filter. The results were as follows: I

Sarnple- Poly-fi-hydroxybutyric acid yield S-4-1 Good 3.6 mg.).

S-4-2 Poor. S-4-3 Good. S-4-4 Good.

S-4-7 Poor. S-4-8 Poor. S-4-13 Good. S-4-16 Good.

0.5 g. of glucose was added to the cultures listed below and the medium was stirred overnight. Another 0.5 g. of glucose was added the following morning. Five hours later each culture was harvested by centrifugation and tested for polyester content in the same manner as above. The amount of product was estimated by the amount of turbidity and theresults wereas follows:

Sample- 1 Poly-p-hydroxybutyric acid yield 5-4-15 Poor S-4-4 I Good S410- Poor S-4-12 Poor In the following example mutants of a species of Rhizobium were prepared. The resulting high density cells were isolated in the same manner described in the previous examples.

Example IV An unidentified specimen of Rhizobium (B-142, Colonial Microbiological Research Institute, Port of Spain, Trinidad) was streaked evenly on three agar plates and allowed to grow for about 24 hours until colonies were just visible.

1 The surface of each agar plate was then dried with glycerin to allow ultraviolet light to penetrate better through the bacteria. This was done by fitting a small piece of filter paper into the inner surface of each Petri dish cover, moistening the paper with a few drops of glycer n and placing the cover over the bacteria for 15-30 minutes. I

The surface of each agar plate was then exposed to ultraviolet light for a particular length of time to produce mutants, e.g. one was exposed for 10 seconds, another for 20 seconds and the third for 60 seconds. The plates were incubated overnight. The 60 second plate was selected as having the highest proportion of mutants since growth Was weakest on this plate indicating, therefore, that most of the original bacteria were killed. The bacteria were scraped from this plate and added to 20 ml. of a sterilized nitrogen-free glucose medium having the following composition:

Nitrogen free mineral solution: 20.0 g. KH PO 37.8 g tleggliiggHeO, 4.0 g. MgSOi. 0.4 g. 09.612, 0.21 g. 'FeCls, 2

Glucose solution: 20.0 1 flushed with nitrogen. g gucose, 500 m1. H20 and The medium was incubated at 30 C. for two hours to promote synthesis of the polyester.

Next, 5 ml. of a sterilized 45% sucrose solution colored pink with safranin were added to each of two plastic centrifuge tubes. 3 ml.'of the bacterial suspension were then added to each tube as a separate layer. Both tubes were centrifuged for 15 minutes at about 512,000 ft./sec.

The procedures described in Example II were followed for aseptically removing samples of the 45% sucrose solution as well as for plating the samples on an agar medium in Petri dishes.

After about 3'days twenty isolated colonies were picked out and grown in ml. batches of the following medium:

Glucose g 40.0 Yeast extract g 1.0 Mineral solution ml 50.0

Mineral solution: 10.0 g. (NH4)2SO4, 10.0 g. KH2PO4. 18.9 g. NaQHPOi-TH O, 2.0 g. MgSOl, 0.2 g. CaClz, 0.06 g. FeCls, 1000.0 ml. H20.

One drop of FeCl ws added to'each 10 ml. batch.

After three days at 30 C. without agitation, each culture was harvested by centrifugation and tested for polyester as in the previous sample. Enough polyester product was obtained sothat it could be filtered and weighed. The results were as follows:

In previous experiments the parent bacterium yielded 1 mg. per ml. of the same medium. The variation in polyester yields in this experiment indicates that these bacteria are actually mutations and not the original bacteria.

In the following experiment a metal centrifuge tube with a permanent siphon was used to separate the bacteria.

Example V An 18 gage (about 1 mm. ID.) stainless steel tube about 9.5 cm. long was soldered onto the inside of a standard metal centrifuge tube so that it extended 0.3 cm. above the top of the centrifuge tube. The top of the 18 gage tube was covered with a gum rubber cap and the centrifuge tube was then autoclaved.

A bacterial suspension was prepared as in Example III. About 30 ml. of a 60% sucrose solution colored pink with safranin was added to the tube and about 5 ml. of the bacterial suspension was added on top of the sucrose solution as a separate layer. The tube was centrifuged for minutes at about 320,000 ft./sec. After centrifugation the rubber cap was pierced with a narrow sterilized syringe needle and about 0.4 ml. of the 60% sucrose solution was withdrawn. The sample was plated and the bacteria were examined, grown and tested for polyester in the usual manner.

I claim:

1. The method of isolating from a suspension of mixed living bacteria the strains therein contained capable of producing the greatest yields of poly-fi-hydroxybutyric acid polyester which comprises treating the suspension with a nutrient to promote the synthesis of the said polyester, adding the thus treated suspension as a separate layer on top of'a layer of a high density solution, centrifuging the resultant combination of layers whereby the more highly-active strains for the production of the said polyester sink into the bottom layer, removing a portion of the bottom layer without mixing it with the layer above, plating the thus removed solution on solid nutrient media, selecting isolated colonies grown on the said media, treating each thus selected strain With a suitable culture medium to promote the said polyester synthesis, harvesting the cells of each culture, drying the harvested bacterial cell mass, dispersing the thus dried cell mass in a poly-B-hydroxybutyric acid solvent, separating the resultant solution of polyester from the cell residue, and recovering and measuring the yield of polyester product from the solvent, whereby the ability of each strain to produce polyester is established by the respective yields of the said polyester.

2. The method according to claim 1 in which the suspension of mixed bacteria is prepared by adding the sample of mixed bacteria to a portion of the following sterilized nitrogen-free mineral solution:

Phosphate butler ml 20.0 MgSO g 1.0 CaCl; g 0.1 FeCl g 0.1 Water l 1.0

Phosphate buffer: 46.0 g. KH2PO4, 86.4 g. Na-2HPO4-7H O, 2000 ml. H2O.

then adjusting the pH of 4-5 with dilute HCl, treating the bacterial suspension with a suitable nutrient to promote the said polyester synthesis, adding a portion of the bacterial suspension as a separate layer to the top of a layer of sterilized 60% sucrose solution.

3. The method of isolating from a suspension of mixed living bacteria the strains therein contained capable of producing the greatest yields of poly-fi-hydroxybutyric acid polyester which comprises treating the suspension with a nutrient to promote the synthesis of the said polyester, adding the thus treated suspension as a separate layer on top of a layer of a high density solution, centrifuging the resultant combination of layers whereby the more highly active strains for the production of the said polyester sink into the bottom layer, siphoning off the upper layer, recovering the high density solution, plating the thus recovered high density solution on solid nutrient media, selecting isolated colonies grown on the said media, treating each thus selected strain with a suitable culture medium to promote the said polyester synthesis, harvesting the cells of each culture, drying the harvested bacterial cell mass, dispersing the thus dried cell mass in a poly-B-hydroxybutyric acid solvent, separating the resultant solution of polyester from the cell residue, and recovering and measuring the yield of polyester product from the solvent, whereby the ability of each strain to produce polyester is established by the respective yields of the said polyester.

4. The method according to claim 3 wherein a sample of soil serves as a source of bacteria.

5. The method according to claim 3 wherein a mixture of mutants prepared by exposing the parent bacterium to ultra-violet light serves as the source of the suspension of bacteria.

6. The method according to claim 3 wherein the high density solution contains sucrose.

7. A method for separating strains of living bacteria capable of synthesizing poly-,B-hydroxybutyric acid from mixtures thereof with other strains contained in aqueous suspension which comprises adding a layer of said suspension on top of an inert solution having a density greater than that of the said suspension and containing a compound sufficiently soluble in water to give a density range for the said solution of about 1.1 to 1.3, centrifuging the resulting combination whereby the cells of bacteria capable of producing said polyester in high yield sink into said solution, and recovering at least a portion of said solution without mixing it with the layer above.

8. The method according to claim 7 wherein the upper layer is siphoned off in order to recover the high density solution.

9. The method according to claim 7 in which the bacterial suspension is treated with nutrient media before centrifugation to promote synthesis of poly-,B-hydroxybutyric acid.

10. The method according to claim 7 in which the bacteria are contained in an aqueous suspension comprised of a mineral solution containing no fixed nitrogen.

11. The method according to claim 7 in which the high density solution contains sugar.

12. The method according to claim 11 in which the sugar is sucrose.

13. The method according to claim 12 in which the density of the sucrose solution ranges from 25 to 60%.

14. The method according to claim 7 in which the high density solution contains a water soluble high polymer.

15. The method according to claim 14 in which the water soluble high polymer is polyethylene glycol.

16. The method according to claim 7 in which the high density solution contains a salt sufiiciently soluble in water to give a density range between about 1.1 to 1.3 and which is non-toxic to bacteria.

17. The method according to claim 7 in which one of the layers is colored to distinguish the separate layers.

18. The method according to claim 7 in which mutants with a high content of poly-,S-hydroxybutyric acid are separated from non-mutated strains containing less of this polyester.

References Cited in the file of this patent Porter: Bacterial Chemistry and Physiology, John Wiley & Sons, Inc., New York, N.Y.,' 1946, pages 97 and 406408.

Lamanna et al.: Basic Bacteriology, The Williams and Wilkins Co., Baltimore, Md., 1953, pages 48-51. 

1. THE METHOD OF ISOLATING FROM A SUSPENSION OF MIXED LIVING BACTERIA THE STRAINS THEREIN CONTAINED CAPABLE OF PRODUCING THE GREATEST YIELDS OF POLY-B-HYDROXYBUTYRIC ACID POLYESTER WHICH COMPRISES TREATING THE SUSPENSION WITH A NUTRIENT TO PROMOTE THE SYNTHESIS OF THE SAID POLYESTER, ADDING THE THUS TREATED SUSPENSION AS A SEPARATE LAYER ON TOP OF A LAYER OF A HIGH DENSITY SOLUTION, CENTRIFUGING THE RESULTANT COMBINATION OF LAYERS WHEREBY THE MORE HIGHLY ACTIVE STRAINS FOR THE PRODUCTION OF THE SAID POLYESTER SINK INTO THE BOTTOM LAYER, REMOVING A PORTION OF THE BOTTOM LAYER WITHOUT MIXING IT WITH THE LAYER ABOVE, PLATING THE THUS REMOVED SOLUTION ON SOLID NUTRIENT MEDIA, SELECTING ISOLATED COLONIES GROWN ON THE SAID MEDIA, TREATING EACH THUS SELECTED STRAIN WITH A SUITABLE CULTURE MEDIUM TO PROMOTE THE SAID POLYESTER SYNTHESIS, HARVESTING THE CELLS OF EACH CULTURE, DRYING THE HARVESTED BACTERIAL CELL MASS, DISPERSING THE THUS DRIED CELL MASS IN A POLY-B-HYDROXYBUTYRIC ACID SOLVENT, SEPARATING THE RESULTANT SOLUTION OF POLYESTER FROM THE CELL RESIDUE, AND RECOVERING AND MEASURING THE YIELD OF POLYESTER PRODUCT FROM THE SOLVENT, WHEREBY THE ABILITY OF EACH STRAIN TO PRODUCE POLYESTER IS ESTABLISHED BY THE RESPECTIVE YIELDS OF THE SAID POLYESTER. 